In light of the safety concerns associated with live vaccines, vaccine formulations are increasingly based on highly purified subunit antigens and/or antigens produced by recombinant DNA technology. However, the poor immunogenicity typical of such antigenic subunits necessitates the use of adjuvants to enhance the immune responses to the protective epitopes in the vaccine. Despite decades of research, very few adjuvants are licensed for use in humans. Unlike live vaccines, current inactivated/subunit vaccines formulated with the licensed adjuvants often confer shorter duration of immunity, largely induce antibody responses, require multiple immunizations to maintain protective immunity, and trigger poor cell-mediated immunity (CMI; TH1 and CD8 T cell memory). Therefore, there is a critical need to identify adjuvants that engender balanced antibody and CMI to protect against both extracellular and intracellular pathogens. Carbomers are polymers of acrylic acid cross-linked with polyalkenyl ethers, and are extensively used as bioadhesives in the pharmaceutical industry and as adjuvants in veterinary and experimental vaccines. We have identified a carbomer-class adjuvant, Adjuplex (ADJ) that elicits potent systemic and mucosal CD4 TH1 and CD8 T cell responses, like a live viral vaccine. In this proposal, as a combination adjuvant, we will leverage the CD4/CD8 T cell responses induced by ADJ to balance and potentiate the immune responses triggered by clinically tested TLR agonists, glucopyranosyl lipid A (GLA) and CpG. Based on preliminary data, we hypothesize that ADJ is an effective antigen delivery system with immune modulating properties. We further hypothesize that combining ADJ with GLA and CpG constitutes a novel adjuvant that couples the effective antigen delivery and immune modulatory properties of ADJ with the immune potentiating effects of GLA and CpG to concomitantly activate multiple innate signaling pathways. In aim 1, we will test whether the combination adjuvant augments the differentiation and persistence of systemic and mucosal tissue-resident memory CD8 T cells along with antibodies and CD4 T cells. Aim 2 will investigate whether and how the combination adjuvant enhances the process of cross presentation by human and murine DCs in vitro. Mechanisms examined will include antigen uptake and targeting to early endosomes, endosomal acidification and retention of antigen, role of lysosomes and proteasome, antigen presentation to nave T cells and the signaling pathways engaged. In aim 3, we will determine whether and how the combination adjuvant enhances mechanisms of cross presentation in vivo. The proposed work has the potential to discover a novel combination adjuvant that will engage multiple signaling pathways in immune cells and harness the unique immune stimulating properties of individual components to engender a balanced and durable humoral and CMI response. The findings from this work will be highly significant and expected to have broad impact on the clinical development of effective vaccines against diverse pathogens.